This invention relates to a strategy for determining the enantiomeric purity of a compound through guest-host complexation, spectroscopy, and chemometric modeling. In particular, this invention relates to the determination of enantiomeric compositions of chiral compounds without regard to whether the concentration of the chiral compound remains constant.
Because of wide differences in the pharmacological and physiological properties of enantiomers, the determination of enantiomeric composition of chiral samples is of considerable interest to chemical research in general and the pharmaceutical industry in particular. In many cases, one enantiomer may be therapeutically active, while the other may be at best, non-active and at worst, toxic or lethal. The need for improved strategies for the assessment of enantiomeric purity arises from increased pressure on the pharmaceutical industry by government agencies for documentation on the pharmacological effects of individual enantiomers and the simultaneous demand in drug development for determination of enantiomeric excess in large combinatorial libraries. While many analytical techniques for chiral analysis have been developed over the years, gas and liquid chromatography, capillary electrophoresis and nuclear magnetic resonance are currently the most widely used. For high throughput screening strategies, slow chromatographic methods are not attractive. Rapid spectroscopic techniques are the most desirable.
Traditional methods of chiral analysis include chiroptical methods, in which the analyte interacts with incident polarized electromagnetic radiation. These include polarimetry, Raman optical activity, and electronic and vibrational circular dichroism. Non-chiroptical methods require some form of chiral auxiliary to interact with the enantiomers forming diastereomers. These include separation techniques, such as chromatography and capillary electrophoresis, NMR, and mass spectrometry.
Chromogenic enantioselective chiral hosts are capable of discriminating between enantiomers of chiral guests through a change in the visible absorption spectrum of the enantioselective complex, i.e., through a color change. (Otagiri, et al., Chem. Pharm. Bull., vol. 23, p. 188, 1975; Schiller, et al., J. Chem. Soc., Faraday Trans., vol. 83, p. 3227, 1987; Park, et al., J. Phys. Chem., vol. 98, p. 6158, 1994; Cox, et al., J. Photochem. Photobiol., vol.39, p. 597, 1984; Bortolus, et al., J. Phys. Chem. A, vol. 106, p. 1686, 2002; Balabai, J. Phys. Chem., vol. 102, p. 9617, 1998). Under this strategy, the complexation of one enantiomer of a chiral substrate with a chiral host results in a visible spectral shift and/or the formation of an entirely new visible band, while little or no color change is observed when the other enantiomer complexes with the chiral host.
Traditonally, cyclodextrins are used as host molecules. Cyclodextrins (“CDs”) are homochiral barrel-shaped sugar molecules that can form transient, non-covalent diastereomeric guest-host complexes with chiral guest molecules. Because the complexes that are formed are diastereomeric, they have different physical properties. Consequently, there are small changes in their spectra. (Suzuki, Electronic Absorption Spectra and Geometry of Organic Molecules, p. 102, 1967). These small spectral variations are often dismissed as having little utility for predicting the composition of a sample because the variations are small, the bands overlap, and the spectra do not appear to show a consistent trend (such as an offset) with composition. However, chemometric methods, such as multivariate regression, offer a variety of powerful techniques for revealing hidden relationships in data that are not immediately apparent.
Multivariate regression modeling (“MRM”) is widely used in chemistry as a means of correlating spectral data with known compositional changes. (Martens, et al., Multivariate Calibration, 1989). While the use of chemometrics in near-infrared spectroscopy is well-established, its use in other spectral regions, such as the ultraviolet region, is not as common. MRM is used for the chemometric analysis of the spectral data of the solutions containing cyclodextrin guest-host inclusion complexes because the solution spectra are composite spectra, simultaneously containing contributions from complexed species (diastereomeric CD inclusion complexes) as well as uncomplexed species that are present because the complexation reaction is not complete.
U.S. Provisional Patent Application No. 60/526,494 pertains to the determination of the enantiomeric composition of various chiral guest molecules by multivariate regression modeling of spectral data obtained from solutions containing cyclodextrin as a chiral auxiliary. The premise behind the approach is that inclusion complex formation between the chiral guest analyte and the homochiral CD host results in the formation of transient diastereomeric inclusion complexes with different physical and spectral properties. As a result, it is observed that, for solutions containing a fixed chiral guest concentration and a fixed CD host concentration, the absorption or emission spectra vary slightly as the enantiomeric composition of the samples is changed. The small spectral variations are then correlated with the known enantiomeric composition of the guest analyte using standard multivariate regression modeling techniques such as partial-least-squares regression (PLS-1). U.S. Provisional Patent Application No. 60/615,123 pertains to a related method for determining enantiomeric purity, but it involves polarimetry as well.
One obvious limitation of the techniques described in U.S. Provisional Patent Applications Nos. 60/526,494 and 60/615,123 is the necessity of keeping the analyte concentration constant. This unrealistic constraint severely limits the usefulness of the technique, particularly in industry, where analyte concentration is likely to fluctuate around an average value.